A novel sulfurizing reagent was developed to facilitate the automated preparation of oligodeoxyribonucleoside phosphorothioates via the standard "phosphoroamidite" approach. These oligonucleotide analogues have been reported to inhibit gene expression in cell cultures. Several polysulfides were tested as potential sulfur-transfer reagents. Of these, the thiosulfonate 3H-1,2-benzothiole-3-one 1,1-dioxide was particularly efficient. A 0.05M solution of the reagent in acetonitrile, converted a dinucleoside monophosphite triester into the corresponding phosphorothioate within 30 sec in near quantitative yields. This reagent led to rapid, efficient (stepwise yields of 99%) and reliable synthesis of phosphorothioate oligomers (15-28 mers) carrying either exclusively or a predetermined number of P(S) linkages without detectable nucleosidic modification. Moreover, the P-31 NMR analysis of an oligomer having exclusively P(S) linkages (24-mer) revealed the presence of-'only a small percentage of P(O) linkages (less than 1%). It has been further demonstrated that the presence of P(O) linkages (ca. 24) within a phorphorothioate oligomer did not alter its effectiveness against the expression of the myc gene product in peripheral blood mononuclear cells relative to the same oligomer that is free of P(O) linkages. The solubility of the above thiosulfonate in various organic solvents in addition to its stability in acetonitrile solution for a prolonged period of time (at least 1 month) without losing efficiency provide a facile access to phosphorothioate oligonucleotides to many laboratories and, thus, add a new dimension to the significance of this work.